Hi,
I am new to RNA-Seq. I have a question when I am choosing library type.
As I know that there are three library types (fr-unstranded, fr-firststrand, fr-secondstrand).
However, what confuses me is that, description of the experiment below contains both 1st strand and 2nd strand.It seems that sequencing was done twice, once in firststrand and once in secondstrand. So I do not know which library type to choose.
Could anyone give me some advice and idea?
Thank you in advance!
supplemental information:
Sequencing instrument is: 454 GS FLX
Strategy: FL-cDNA
Layout is single-end
Data is here: https://www.ncbi.nlm.nih.gov/sra/SRX015663[accn]
There are three parts in experiment design:
cDNA production (1st strand)
First-strand cDNA poly(A) synthesis was performed using the SuperScript III First-Strand kit (18080-051, Invitrogen). One microgram of total RNA was reverse-transcribed using the oligo(dT)20 primer provided by the kit in a reaction volume of 20 µL. A control reaction was also performed without the Superscript reverse transcriptase enzyme to monitor genomic contaminations.
cDNA production (2nd strand)
Full-length cDNA synthesis First-strand cDNA synthesis was performed with the PrimeScript Reverse Transcriptase (Clontech Laboratries) using the SMART (Switching Mechanism at 5' end of RNA Transcript) PCR technology (Clontech Laboratories), but following a modified protocol suggested by Roche Diagnostics to optimize sequencing using the 454 FLX Sequencing technology. Total RNA was reverse-transcribed using a modified CDS III oligo(dT) (Invitrogen Life Technologies): 5'-xxxxx-3' (N corresponds to the mix of A, G, or C). At the 5'-terminus of the template, a poly(C) tail is added to the cDNA using the terminal transferase activity of the reverse transcriptase. The SMART V oligonucleotide provided with the kit hybridizes to the poly(C) tail to form an RNA/DNA hybrid: 5'-xxxxxxx-3'.
full length LD PCR
For the LD PCR reaction, we used the Advantage 2 PCR Kit (Clontech Laboratries). Only sscDNAs that have the 5'-SMART anchor can be used as a template for the LD PCR reaction, thus ruling out eventual genomic DNA contamination.
